Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7944-7948, September 1992 Biochemistry Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol (disulfide reduction/thiol oxidation/redox potential) DAVID M. ROTHWARF AND HAROLD A. SCHERAGA* Baker Laboratory of Chemistry, Cornell University, Ithaca, NY 14853-1301 Contributed by Harold A. Scheraga, June 15, 1992

ABSTRACT The equilibrium and rate constants for the KObs [DTToX] [GSH]2 reaction between oxidized and reduced glutathione and oxi- eq [Drrred] [GSSG]' [2] dized and reduced dithiothreitol have been determined at several pH values and temperatures. The measurements in- where the brackets indicate concentrations at equilibrium. volve approach to equilibrium from both directions, quenching Unfortunately, a wide range of values has been reported of the reaction by lowering the pH or by addition of methyl for this thiol-disulfide exchange equilibrium constant. Values methanethiosulfonate, separation of reactants and products by of 8800 M at pH 7 and 300C (10) and 13,000 M at pH 7 and an reverse-phase HPLC, and determination of their concentra- unspecified temperature (11) have been reported, based on an tions. Analysis of reaction mixtures was carried out at various indirect technique involving a lipoamide dehydrogenase- times to assure that equilibrium had been reached and to mediated reaction between NAD+ and lipoamide. Use of determine kinetic constants prior to the attainment of equilib- intermediates populated during the regeneration of bovine rium. pancreatic trypsin inhibitor (BPTI) led to a value of 1200 M at pH 8.7 and 250C (12). A direct HPLC method, very similar The thiol-disulfide interchange reaction is important in a to the technique to be reported here, gave values of -200 M variety of biological systems (1-3) and, in particular, in at pH 7 and 8, 250C, and 380 M at pH 8.7, 250C (13). In the studies of the regeneration of disuffide-containing proteins direct method (13), the equilibrium was approached from from their reduced forms (4-6). The thiol/disulfide reagents only one direction and insufficient experimental details were in widest use in studies of the regeneration of proteins are provided to resolve the controversy. Our own indirect mea- oxidized glutathione (GSSG) and reduced glutathione (GSH), surements (unpublished results), obtained while studying the primarily because they are known to occur at significant regeneration of ribonuclease A, resulted in a value of 260 M concentrations in biological systems (7) and because they at pH 8, 25°C, and are in agreement with those of Chau and exhibit a suitable redox potential. Nelson (13). The oxidation of monothiols such as GSH is a bimolecular While it will not be discussed in detail here, the value ofthis process and is entropically different from the unimolecular equilibrium constant and the rate of reduction of GSSG by process of disulfide formation that occurs in proteins. In dithiothreitol (DTTmd) are essential for explaining the path- contrast, the formation of cyclic disulfides such as oxidized ways ofregeneration ofribonuclease A and for understanding dithiothreitol (DTToX) results from a unimolecular process. the general problem of how native protein structures regen- Glutathione forms stable mixed disulfides with protein thiols, erate. The purpose of this paper is to present experimental which greatly complicates studies of regeneration pathways. details of the direct method, sufficient to confirm the accu- On the other hand, while DTTOX is a much weaker oxidizing racy of the results of Chau and Nelson (13) at pH 8 and 25°C, agent, it does not form stable mixed disulfides (because the which are consistent with our data from the regeneration of cyclization reaction is very fast), making it a useful reagent ribonuclease A, and to determine the values of the equilib- for studies of protein regeneration (5, 8). It has been argued, rium constants and rate constants under the solution condi- though incorrectly, that, while these reagents differ in their tions that are directly relevant to our work on the regener- entropies of disulfide formation, they are similar in their ation of ribonuclease A (8). We have also carried out equil- abilities to regenerate protein when concentrations are ibrations under other conditions (pH 7, 30°C) to permit direct adjusted to give similar redox potentials (9). comparison with the values for the equilibrium and kinetic The role that these different types (mono- and cyclic thiol) constants determined by Szajewski and Whitesides (10). of reagents play is a key feature of the detailed regeneration processes of proteins. Any attempt to distinguish these different roles requires a quantitative determination of their MATERIALS AND METHODS relative redox potentials. In order to make comparisons Materials. Ultrapure DTTred was obtained from Boeh- between the results ofregeneration experiments using dithio- ringer Mannheim and recrystallized from absolute ether. threitol and those using glutathione, it is necessary to know DTTOX (Sigma) was purified by the method ofCreighton (14). the equilibrium constant for the reaction between glutathione GSH and GSSG were obtained from Boehringer Mannheim and dithiothreitol. The equations describing this equilibrium and were purified by reverse-phase HPLC on a 5-pm C18 are Dynamax column (Rainin, Woburn, MA) with 0.09%o tri- fluoroacetic acid (TFA) in water isocratically as the mobile GSSG + DTTred ; 2GSH + DTfoX [1] Abbreviations: DTTOX, oxidized dithiothreitol; DTTed, dithiothrei- tol; GSH, reduced glutathione; GSSG, oxidized glutathione; BPTI, The publication costs of this article were defrayed in part by page charge bovine pancreatic trypsin inhibitor; DDS, disulfide detection system; payment. This article must therefore be hereby marked "advertisement" MMTS, methyl methanethiosulfonate; TFA, trifluoroacetic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7944 Downloaded by guest on September 24, 2021 Biochemistry: Rothwarf and Scheraga Proc. Natl. Acad. Sci. USA 89 (1992) 7945 phase. All other reagents were of the highest grade commer- alent volume of a TFA solution sufficient to lower the pH to cially available. 2. The concentration of TFA used was in the range 150-300 Purity. Because the equilibrium constant of Eq. 2 is mM. In other experiments, a 25-fold volume of 25 mM TFA expected to be large and has a molar concentration depen- was used to lower the pH to 2. There was no difference within dence, the concentrations of GSH and DTTYX at equilibrium experimental error between the two techniques. While it is will be as much as 4 orders of magnitude greater than those unlikely that different acid concentrations during quenching of GSSG and DTTmd under the conditions used here. There- could yield the same equilibrium constant if shuffling oc- fore, even a small level of impurity could lead to erroneous curred during the quenching, it is not a definitive control. results if it were coeluted with DTTr"d or GSSG. All reagents Therefore, an additional method was used to quench some of were, therefore, purified to >99.9% purity as judged by the samples. A 15-fold molar excess of methyl methanethio- HPLC at 210 nm. In addition, the GSSG and GSH were sulfonate (MMTS) over free thiol was added in a 3:1 volume checked by NMR on a Varian XL-400 instrument prior to ratio at pH 8.0. After 5 min, the blocking reaction was purification, because we have observed that glutathiones quenched with 0.5 M TFA to pH 2 prior to HPLC analysis. obtained from some suppliers contained significant amounts Within experimental error, there was no difference between (in some cases >20%) of a-glutamylcysteinylglycine (M. the results from any of the quenching procedures. In control Adler, D.M.R., and H.A.S., unpublished results) as well as experiments on mixtures of DTTJX and DTTred, the MMTS cysteinylglycine (T. W. Thannhauser and H.A.S., unpub- quenching conditions blocked >95% of the DTTred com- lished results). Significant heterogeneity in the GSSG and pletely (the remainder presumably cyclized to DTTOX before GSH may be the origin of the wide range of values reported the second thiol of DTTred was blocked). for the dithiothreitol-glutathione equilibrium constant. NMR Fractionation of Species. After quenching, all equilibration spectra of the materials used in these studies revealed only were on a Waters radial the presence of GSSG and GSH. mixtures fractionated Nova-Pak C18 The high purity of the starting materials, however, is an compression column. A Spectra-Physics SP8800 gradient inadequate control to exclude experimental errors due to pump and a Gilson UV116 detector set to 210 nm constituted contamination, since the formation of small amounts of the delivery and detection system. A binary gradient using degraded starting material could also complicate the inter- solvent systems A (0.09% TFA/water) and B (0.09%o TFA/ pretation of the data if they were coeluted with DTTred or acetonitrile) was run at a flow rate of 1 ml/min. The com- GSSG. Therefore, we measured the equilibrium constant position of the mobile phase was held at 100% A for 10 min, over a >10-fold range of the starting thiol and disulfide and then a linear gradient to 15% B in 30 min was imple- concentrations. Since the equilibrium constant has a molar mented. This gradient was used in all subsequent HPLC concentration dependence, a 10-fold increase in the concen- analyses described below. All data were digitized and stored trations of both GSH and DTTYX will lead to an -1000-fold on a Prime 750 computer. A typical chromatogram is shown increase in the product of the concentrations of GSSG and in Fig. 1. DTTred. Since it is the concentrations of GSSG and DTTred Relative Extinction Coefficients. Given the form of the that are sensitive to the level of impurities, and since they equilibrium expression as shown in Eq. 2, it followed that the increase nonlinearly with increases in the total concentration minimum error could be obtained by use ofrelative extinction of material added, the consistency of K~bS serves as an coefficients of the reactants and products, since this would indicator for the absence of impurities. We have exploited not require a direct measurement of the individual concen- this nonlinear relationship further by varying the concentra- trations of each species. The disulfide detection system tions of reagents such that either [DTTred] >> [GSSG] or (DDS) of Thannhauser et al. (15) was used, but with the [GSSG] >> [DTTred] at equilibrium. We also carried out addition of a Lee mini-inert solenoid valve interfaced to a equilibrations over a range of times from 10 to 3300 min. home-built digital control box that permitted accurate split- While some small uncharacterized peaks were observable in ting ofthe column effluent without loss ofresolution. Seventy the chromatograms from the longer-time equilibrations, the measured equilibrium constant was unaffected by their for- 0.5 mation. This indicates that no significant population of deg- GSH radation products was coeluted with DTTred or GSSG. It should be noted that any small level of impurities that are 0.4 coeluted with DTTIX or GSH will not have any significant E effect on the measured equilibrium constant. 0 Equilibration. Equilibrations were carried out in Pierce 0.3 Reactivials or in screw-cap tubes with septum tops. They DTT red were placed in a jacketed bath connected to a Haake F-1 circulating bath with tap water cooling or to a Fisher Isotemp 0.2 GSSG refrigerated circulator. Temperature regulation was better 0 ~~~~~~~~~~~DTTO than +0.20C. Samples were kept under a constant stream of humidified argon. Buffer containing 100 mM Tris and 2 mM 0.1 EDTA, which had been sparged with argon, was used for the equilibrations at pH 7.5, 8.0, and 8.7, and the pH was adjusted at the temperature used. Equilibrations at pH 7.0 were carried out in 100 mM phosphate for consistency with 0 10 20 30 the solution conditions used in earlier studies (10, 11). The pH Time (min) of each sample was checked on a Radiometer PHM64 re- search meter with an microelectrode and FIG. 1. HPLC chromatogram of an equilibrated mixture ofGSH, pH Ingold adjusted an of the addition of NaOH HCO to maintain the desired DTTred, GSSG, and DTTOX quenched with equivalent volume by and/or 250 mM TFA. The DTTred and GSSG peaks are also shown expanded pH. by a factor of 20. Chromatographic conditions are described in the Quenching. Since it was crucial to ascertain that the text. The sample corresponds to a reaction time of 1100 min at pH equilibrium was not perturbed when the reaction was 8.0, 250C. The concentrations of each species at this time were as stopped, several different quenching methods were em- follows: GSH, 1.39 x 10-2 M; DTTred, 4.03 x 10-4 M; GSSG, 1.90 ployed. Acid quenching was carried out by adding an equiv- x 10-s M; and DTTOX, 7.69 x 1O-3 M. Downloaded by guest on September 24, 2021 7946 Biochemistry: Rothwarf and Scheraga Proc. Nad. Acad. Sci. USA 89 (1992)

percent of the column effluent was directed toward a Gilson surements were made at 412 nm and 220C on a modified Cary UV116 detector set to 210 nm (to provide a measure of solute 14 spectrophotometer (16). The standard deviation of the concentrations), and the remaining 30%o was directed to the measurements was less than the 3% uncertainty in the DDS (operating at 412 nm to provide a measure of thiol and extinction coefficient of 2-nitro-5-thiobenzoic acid (17). We disulfide concentrations) at a switching speed of 2 cycles per have used a value of 13,900 M'1cm-1 for the extinction second. The DDS has been shown to be quantitative with a coefficient ofthe dianion of2-nitro-5-thiobenzoic acid, which linear response over the range of 10-10 to 10-12 mol of thiol was determined in our laboratory from studies ofthe 2-nitro- or disulfide (15), and our experiments were carried out within 5-thiosulfobenzoic acid reagent (17). Other values reported in the linear range. All the data were digitized and stored on a the literature, 13,600 M~-1cm-1 (18, 19), 13,700 M-1 cm-1 Prime 750 computer. Peak areas were determined from the (20), and 14,150 M-1 cm'1 (13, 21), are well within the stated digitized data, and relative concentrations were determined 3% standard deviation (at 99% confidence) of our value (17). for GSSG, DTTred, and GSH from the DDS areas. The The concentrations of the four species in the equilibrium relative extinction coefficients, expressed with respect to mixture were determined from the total thiol concentration GSH, were calculated from the measured peak areas at 210 and the relative extinction coefficients. The minimum uncer- nm. tainty in the equilibrium constants at 95% confidence is 8.5%, Unfortunately, DTTOX is not reduced by the sulfite com- which arises from the uncertainties in the relative extinction ponent of the DDS under the conditions used and, therefore, coefficients and the extinction coefficient of 2-nitro-5- does not react with 2-nitro-5-thiosulfobenzoic acid. The thiobenzoic acid. relative extinction coefficient of DTTX with respect to Kinetics. By taking aliquots ofthe reaction mixtures prior to DTTrd was, therefore, determined in a different manner. the attainment of equilibrium, and taking account of the DTTrd was dissolved in 100 mM (pH an Tris 8.0), and forward and reverse reactions (by expressing kr as kftIK2b), amount of phenylalanine, an internal standard, was added we have been able to determine the apparent forward rate sufficient to give an integrated area relative to DTTred at 210 the nm of between 0.2 and 0.6. Air was not excluded, so that constant, kf) S, for dithiothreitol-glutathione equilibrium 1. oxidation could take place. At various times over a period of process shown in Eq. Since no starting mixture contained 24 hr, aliquots were removed, the pH was lowered to 2 by the both DTT"' and GSH, the starting concentration ofthe other addition of 0.5 M TFA, and the solution was analyzed by three species could be determined. The data were then fit with reverse-phase HPLC using detection at 210 nm. The ratio of the aid of a simple Runge-Kutta program to integrate the rate the gain in peak area ofDTTOX divided by the loss in peak area equations numerically (22), using the experimentally deter- of DTTred gave the relative extinction coefficient for DTPTX mined mass quotient, ([GSH2 [DTTOx])/([GSSG] [DTTId]), with respect to DTTed. The ratio of the areas of the phenyl- as the target function. The program was run on a Prime 750 alanine peaks at zero time and at subsequent times was used computer. to normalize the results. To check the validity of this method and to show the consistency of the two methods used to RESULTS determine relative extinction coefficients, the same process was carried out by using GSH instead ofDTTred and allowing Data obtained at pH 8.0, 250C, are shown in Table 1. The it to oxidize slowly in air. There was no difference within agreement ofthe experimentally determined equilibrium con- experimental error between the values obtained by the two stant over a 30-fold range of concentration when approached techniques. The standard deviation of the mean (95% confi- from either direction, as well as the consistency ofthe results dence limit) for the relative extinction coefficients was <2% with different blocking methods, indicates the absence ofany in all cases. The DDS technique was used to determine the significant experimental artifacts. In addition, the consistent relative extinction coefficients for GSH and DTTred that had value determined for the observed rate constant for the been blocked with MMTS, because they have different reduction ofGSSG by DTTrd, when the reaction was started extinction coefficients than the unblocked forms. from either direction, as shown in Table 1, further supports The linear response of the UV detection system was the accuracy of the experimental techniques used. The com- checked by running a series of calibration curves for each of plete set of data under all the solution conditions used is the five components, GSSG, GSH, DTTJX, DTTred, and summarized in Table 2. All rate constants and equilibrium phenylalanine, over 4 orders of magnitude. While some constants presented were derived from data obtained by nonlinearity was observed for some species at the extremes approaching equilibrium from both directions. Comparison of concentration, the response for all components was linear (correlation coefficient, >0.999) over a range of -3 orders of Table 1. Glutathione-dithiothreitol data at pH 8.0, 25°C magnitude of the integrated area. Equilibration experiments Initial concentration, mM Kobs ks in which all four ofthe components fell within the linear range were the only ones used. This placed restrictions on the range GSH DTTOx DTTred GSSG M minM of concentrations that could be used in these experiments. 0 0 1.06 1.28 186 Starting concentrations were selected so as to increase the 0 0 1.44 1.32 190 likelihood that the concentration of each species would fall in 0 0.03 8.15 8.42 231 202 the desired range. In some cases, it was necessary to run two 0 0.26 3.77 4.23 237 chromatograms with very different injection volumes so that 0 0.30 4.04 5.21 170 all species were in the linear range in at least one chromato- 0 0.73 7.37 13.9 1940 - gram. In these cases, at least one component was kept in the 1.11 1.81 0 0 235 linear range in both chromatograms to serve as a standard for 9.89 8.85 0 0.04 225 180 scaling. 13.1 31.0 0 0.21 235 - Equilibrium Constant. As the first step in determining the 14.3 28.1 0 0.04 235 equilibrium constant, the total thiol concentration was ob- 14.7 29.9 0 0.23 243t tained in separate quadruplicate measurements using Ell- All reaction mixtures were quenched with acid except those man s reagent, 5,5'-dithiobis(2-nitrobenzoic acid). All vol- indicated otherwise. ume measurements in this investigation were made with *Observed rate constant for the reduction of GSSG by DTTrd. Hamilton 1000 series Gas-Tite syringes or Finnpipette mi- t213 M with MMTS blocking. cropipettors, which were calibrated before use. Ellman mea- *249 M with MMTS blocking. Downloaded by guest on September 24, 2021 Biochemistry: Rothwarf and Scheraga Proc. Natl. Acad. Sci. USA 89 (1992) 7947 Table 2. Summary of glutathione-dithiothreitol equilibrium and kinetic data Temp., KebS x KS- x 10-4 kA~bs X 10-2, kf X 10-3, pH C 10-2, M M min-1 M-l min-'.M-1 7.0 30 1.84 + 0.16 1.29 0.354 + 0.081 5.64 7.5 25 1.99 ± 0.20 1.31 0.61 ± 0.10 3.1 8.0 15 2.21 ± 0.26 1.20 8.0 25 2.29 ± 0.20 1.24 1.86 ± 0.16 3.09 8.0 37 2.32 ± 0.23 1.26 4.8 ± 1.3 8.0 8.7 25 3.75 ± 0.32 0.945 7.6 + 1.5 2.9 Errors defined 95% confidence limits. K~bS is given by Eq. 2; Ks- is given by Eq. 3; k4bs is the observed rate constant for the reduction of GSSG by DTTred; and kf is given by Eq. 5.

of the equilibrium constants obtained at different pH values (1 + + = Obs 1opK2-pH loPKl+pK2-2pH) can be made by comparing the values of KS-: kfkf [5] (2 + lopK2-pH) [GS ]2 [DTTOx] The values of kf are shown in the last column ofTable 2. The [GSSG] [DTT(-S )2] values at 250C are in excellent agreement with one another. Temperature Dependence. Measurements were made at pH This equilibrium constant should be independent of pH. 8.0 and 150, 250, and 37°C because of their relevance to our However, since the concentrations of the thiolate anions are studies ofthe refolding ofribonuclease A. As shown in Table not measured directly, it is necessary to calculate them by 2, there is no difference within the experimental error be- using the known pK values of the thiols involved. The tween the measured equilibrium constants as a function of expression for KS- as a function of KIbS is as follows: temperature in this temperature range.

KS-= (1 + + KObS 1OpK2-pH lOPKl+PK2-2pH) 1 [4] DISCUSSION (1 + JOPKGSH-pH)2 While the equilibrium constants reported here are in excellent where pKGSH refers to the dissociation constant for the thiol agreement with the values reported by Chau and Nelson (13), of GSH, and pK, and pK2 refer to the first and second it appears that our data are in conflict with the results dissociation constants for the first and second thiols of obtained in earlier studies in which the indirect lipoamide DTTred, respectively. We have used 9.2 and 10.1 for the dehydrogenase-mediated reaction system was used. This is values of pK, and pK2, respectively (20), and 8.72 for the especially perplexing because the lipoamide dehydrogenase- value of pKGSH (23), all at 30°C. mediated method appears to give more consistent results As shown in Table 2, the value of Ks- at pH 8.7 does not when applied to cyclic disulfide reagents (27). In addition, an agree with the value ofKS- determined at the other pH values experimental check on the value of the dithiothreitol- within the experimental error. This could be the result of lipoamide equilibrium constant by determining the concen- differences in pK as a function of temperature, since the pK tration of species by UV absorbance measurements (10) is in values that we used were determined at 30°C, or some excellent agreement with the value obtained by the lipoamide systematic error that we have neglected to take into account. dehydrogenase-mediated method (10). However, the more likely explanation is that it is the result A similar inconsistency has been found between the values of in the pK values of GSH and ofthe equilibrium constant inferred for the mercaptoethanol- experimental uncertainty when DTTrd. In particular, an increase in the pK of GSH of <0.1 dithiothreitol and glutathione-dithiothreitol reactions unit would result in the agreement ofall the KS- values within based on separate measurements using the indirect lipoamide reaction system and the values experimental error. The reported values for the pK of GSH dehydrogenase-mediated measured a more direct NMR assay (G. M. Whitesides, vary (23), we have used here, to 9.2 at 25°C by from 8.72 which personal communication). This suggests that the problem (24). Intermediate values have also been reported at 25°C may be a general one involving monothiols when the indirect (25). A further complication is that the ionization ofthe amino method is used. Since the equilibrium is approached by group of GSH has been shown to affect the observed pK of starting with GSH, inhibition or inactivation ofthe lipoamide the thiol (25). Since the amino group has apK of -9.1 (24, 25) dehydrogenase prior to the attainment of equilibrium would and would be significantly deionized at pH 8.7, a local lead to a value of the apparent equilibrium constant that is electrostatic effect from the amino group could explain the substantially larger than the actual one. disparity at this pH. There are several possible explanations for the difference Kinetics. The fifth column of Table 2 shows the experi- between the equilibrium constant of 375 M that we measured mentally determined values of 1fObs, the observed rate con- at pH 8.7, 250C, and the value of 1200 M obtained by stant for the reduction of GSSG by DTTr,5d. Comparison of Creighton and Goldenberg (12). The most obvious one is that - our rate constant at pH 7.0, 30°C (35.4 ± 8.1 min M-1), with the interaction with BPTI is different for dithiothreitol and that of Szajewski and Whitesides (14.1 2.1 min-1 M-1) (10) glutathione-i.e., that there are specific interactions between indicates that these values do not agree within experimental BPTI intermediates and either or both of the redox reagents. error. The only other published value for the reduction of An additional and equally plausible explanation is that the GSSG by DTTred is 810 min-1-M-1 at pH 8.7 and 25°C (read difference is due to experimental limitations of the method from figure 5 in ref. 26), which agrees within experimental used by Creighton and Goldenberg. The determination ofthe error with our value of 760 + 150 min-1 M-1. The values of glutathione-dithiothreitol equilibrium constant using BPTI the rate constants at different pH values are more easily intermediates was made from kinetic data and not under compared by considering kf, which is the rate constant for the conditions in which an equilibrium or preequilibrium condi- reduction of GSSG by the thiolate anion of DTTred. The tion prevailed. As a result, the determination of the rate pH-independent rate constant kf is related to Afbs by the constants was dependent on the accuracy of the model to following expression: which the data were fit. Since our value and that obtained by Downloaded by guest on September 24, 2021 7948 Biochemistry: Rothwarf and Scheraga Proc. Nad. Acad. Sci. USA 89 (1992) Creighton and Goldenberg differ by only a factor of 3, it 9. Wearne, S. J. & Creighton, T. E. (1988) Proteins: Struct. would require an error of only S5O% in their forward and Funct. Genet. 4, 251-261. reverse rate constants to reconcile the two results. Further- 10. Szajewski, R. P. & Whitesides, G. M. (1980) J. Am. Chem. more, recent studies ofthe regeneration ofBPTI (28) indicate Soc. 102, 2011-2026. that the quenching technique used in ref. 12 (iodoacetate 11. Cleland, W. W. (1964) Biochemistry 3, 480-482. 12. Creighton, T. E. & Goldenberg, D. P. (1984) J. Mol. Biol. 179, blocking) was inadequate and that significant rearrangement 497-526. occurred during the blocking. Hence, the distribution of 13. Chau, M.-H. & Nelson, J. W. (1991) FEBS Lett. 291, 2%-298. intermediates that was measured did not represent the true 14. Creighton, T. E. (1977) J. Mol. Biol. 113, 295-312. distribution before blocking. 15. Thannhauser, T. W., McWherter, C. A. & Scheraga, H. A. In conclusion, the data of Table 2 indicate that DTTOX is a (1985) Anal. Biochem. 149, 322-330. strong enough oxidizing agent for regenerating disulfide- 16. Denton, J. B., Konishi, Y. & Scheraga, H. A. (1982) Biochem- containing proteins from their reduced forms. They are being istry 21, 5155-5163. used in our analysis of regeneration data for ribonuclease A, 17. Thannhauser, T. W., Konishi, Y. & Scheraga, H. A. (1984) a preliminary communication of which has already appeared Anal. Biochem. 138, 181-188. 18. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. (8). 19. Danehy, J. P., Elia, V. J. & Lavelle, C. J. (1971) J. Org. Chem. This research was supported by Grant GM14312 from the National 36, 1003-1005. Institute of General Medical Sciences of the National Institutes of 20. Whitesides, G. M., Lilburn, J. E. & Szajewski, R. P. (1977) J. Health. Support was also received from the National Foundation for Org. Chem. 42, 332-338. Cancer Research and from the Cornell Biotechnology Program. 21. Riddles, P. W., Blakeley, R. L. & Zerner, B. (1979) Anal. Biochem. 94, 75-81. 1. Ziegler, D. M. (1985) Annu. Rev. Biochem. 54, 305-329. 22. Wiberg, K. B. (1986) in Techniques ofChemistry, Investigation 2. Gilbert, H. F. 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